Combination of an oxidant and a photoactivator for the healing of wounds

ABSTRACT

There is provided a wound healing composition which comprises at least one oxidant, at least one photoactivator capable of activating the oxidant and at least one healing factor chosen from hyaluronic acid, glucosamine and allantoin in association with a pharmaceutically acceptable carrier. In addition, a method of topically treating wounds using at least one oxidant and at least one photoactivator capable of activating the oxidant followed by illumination of said photosensitizer is disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/127,435, filed on May 3, 2011, which is a national stage filing under35.U.S.C. §371 of International Application No. PCT/CA2009/001608, filedon Nov. 6, 2009, which claims the benefit of the filing date under 35U.S.C. §119(e) to U.S. Provisional Application 61/112,235, filed on Nov.7, 2008, the entire content of which is hereby incorporated byreference. International Application No. PCT/CA2009/001608 was publishedunder PCT Article 21(2) in English.

BACKGROUND

(a) Field

The subject matter disclosed generally relates to a wound healingcomposition and method of treatments thereof.

(b) Related Prior Art

The process of wound repair is fundamental to the restoration of tissueintegrity and function after operative procedures or traumatic injury.Delayed wound healing and dehiscence of operative wounds represent asignificant clinical problem.

Photodynamic therapy using photoactive dyes such as Erythrosine B,Safranin O has been employed to destroy bacteria, as described in WO05/032459 and WO 05/021094, both to Albrecht et al. The photoactive dyesare employed to directly destroy bacteria. The compositions described inthese patent applications lack oxidants and healing factors, and theyare not employed for directly promoting wound healing.

U.S. Pat. No. 6,056,548 to Neuberger et al. describes a method ofdestroying bacteria in the oral cavity, and promotes bucal hygiene usingphotoactive dyes. This patent also describes using a bleaching agent,hydrogen peroxide, to photobleach and destroy the photoactive dye usedfor destroying bacteria. However, the compositions used do not mentionhealing factors and they are not employed for directly promoting woundhealing.

WO 08/013962 to Grafe et al. describe the use of a composition whichcomprises collagen and a photoactivatable molecule, temoporfin (mTHPC)for the in vivo cross linking of collagen to strengthen and stabilizethe microstructure of a collagen scaffold. This patent also describesthat composition displayed anti-microbial effect, and disinfects thetreatment site and curbs microbial growth. However, these compositionsdo not contain oxidants, or healing factors and therefore promote woundhealing by strengthening the collagen scaffold formed and bacteriadestruction.

Although destroying bacteria present in a wounded site is conducive towound healing, it does not directly stimulate wound repair. Therefore,it would be highly desirable to be provided with a novel composition forthe healing of skin damages and wounds in order to not only destroybacteria, but also to improve and accelerate the healing processfollowing the establishment of pathologic lesions, trauma or injury.

SUMMARY

In accordance with one embodiment there is disclosed a wound healingcomposition which comprises at least one oxidant, at least onephotoactivator capable of activating the oxidant, and at least onehealing factor chosen from hyaluronic acid, glucosamine and allantoin,in association with a pharmaceutically acceptable carrier.

The oxidants may be chosen from hydrogen peroxide, carbamide peroxideand benzoyl peroxide.

The wound healing composition may further comprise at least onehydrophilic gelling agent.

The hydrophilic gelling agent may be chosen from glucose, modifiedstarch, methyl cellulose, carboxymethyl cellulose, propyl cellulose,hydroxypropyl cellulose, Carbopol® polymers, alginic acid, sodiumalginate, potassium alginate, ammonium alginate, calcium alginate, agar,carrageenan, locust bean gum, pectin, gelatin.

The photoactivators may be chosen from a xanthene derivative dye, an azodye, a biological stain, and a carotenoid.

The xanthene derivative dye may be chosen from a fluorene dye, afluorone dye, and a rhodole dye.

The fluorene dye may be chosen from a pyronine dye and a rhodamine dye.

The pyronine dye may be chosen from pyronine Y and pyronine B.

The rhodamine dye may be chosen from rhodamine B, rhodamine G andrhodamine WT.

The fluorone dye may be chosen from fluorescein and fluoresceinderivatives.

The fluorescein derivative may be chosen from phloxine B, rose bengal,and merbromine.

The fluorescein derivative may be chosen from eosin and erythrosine.

The azo dye may be chosen from methyl violet, neutral red, para red,amaranth, carmoisine, allura red AC, tartrazine, orange G, ponceau 4R,methyl red, and murexide-ammonium purpurate.

The biological stain may be chosen from saffranin O, basic fuchsin, acidfuschin, 3,3′ dihexylocarbocyanine iodide, carminic acid, andindocyanine green.

The carotenoid may be chosen from crocetin, α-crocin(8,8-diapo-8,8-carotenoic acid), zeaxanthine, lycopene, α-carotene,β-carotene, bixin, and fucoxanthine.

The carotenoid may be present in the composition as a mixture chosenfrom saffron red powder, annatto extract and brown algae extract.

The wound healing composition may further comprise at least onechelating agent.

The chelating agent may be chosen from ethylenediaminetetraacetic acid(EDTA) and ethylene glycol tetraacetic acid (EGTA).

The wound healing composition may further comprise at least onelipolysis stimulating factor.

The lipolysis stimulating factor may be chosen from caffeine andparaxanthine.

In accordance with one embodiment, there is disclosed a method for woundhealing which comprises the steps of a) topically applying on apatient's skin a composition comprising at least one oxidant, at leastone photoactivator capable of activating the oxidant; and b) treatingsaid skin of step a) to actinic light for a time sufficient for saidphotoactivator to cause activation of said oxidant.

The method for wound healing may comprise exposing the skin to actiniclight for a period of about 60 seconds to about 5 minutes.

The method for wound healing may comprise exposing the skin to actiniclight for a period of about 60 seconds to about 5 minutes per cm² of anarea to be treated.

The method for wound healing may comprise exposing the skin to a sourceof actinic light that is in continuous motion over the area beingtreated.

The method for wound healing may comprise exposing the skin to actiniclight that may be visible light having a wavelength between 400 nm and600 nm.

The following terms are defined below.

The term “hydrophilic gelling agent” is intended to mean a material thatthickens and stabilizes liquid solutions, emulsions, and suspensions.Hydrophillic gelling agents dissolve in liquid and provide a structuregiving the resulting gel an appearance of a solid matter, while beingmostly composed of a liquid. Hydrophillic gelling agents are verysimilar to thickeners.

The term “actinic light” is intended to mean light energy emitted from aspecific light source (lamp, LED, or laser) and capable of beingabsorbed by matter (e.g. the photoactivator defined below) and producean identifiable or measurable change when it interacts with it; asclinically identifiable change we can presume a change in the color ofthe photoactivator used (e.g. from red to transparent).

The term “photoactivator” is intended to mean a chemical compoundcapable of absorbing actinic light. The photoactivator readily undergoesphotoexcitation and then transfers its energy to other molecules, thusenhancing or accelerating the dispersion of light, and enhancing oractivating the oxidant present in the reaction mixture.

The term “oxidant” is intended to mean a either a chemical compound thatreadily transfers oxygen atoms and oxidize other compounds, or asubstance that gains electrons in a redox chemical reaction.

The term “chelating agent” is intended to mean a chemical that removesmetal ions, such as iron, and holds them in solution.

The term “healing factor” is intended to mean a compound that promotesor enhances the healing or regenerative process of a tissue.

The term “lipolysis” is intended to mean the process in which lipids arebroken down into their constituent fatty acids.

The term “time of exposure to actinic light” is intended to mean thetime a tissue, skin or wound is exposed to actinic light per applicationof actinic light.

The term “total time of exposure to actinic light” is intended to meanthe cumulative time a tissue, skin or wound is exposed to actinic lightafter several application of actinic light.

The term “pharmaceutically acceptable carrier” is intended to mean apreservative solution, a saline solution, an isotonic (about 0.9%)saline solution, or about a 5% albumin solution, suspension, sterilewater, phosphate buffered saline, and the like. Other buffering agents,dispersing agents, and inert non-toxic substances suitable for deliveryto a patient may be included in the compositions of the presentinvention. The compositions may be solutions, suspensions or anyappropriate formulation suitable for administration, and are typicallysterile and free of undesirable particulate matter. The compositions maybe sterilized by conventional sterilization techniques

Features and advantages of the subject matter hereof will become moreapparent in light of the following detailed description of selectedembodiments, as illustrated in the accompanying figures. As will berealized, the subject matter disclosed and claimed is capable ofmodifications in various respects, all without departing from the scopeof the claims. Accordingly, the drawings and the description are to beregarded as illustrative in nature, and not as restrictive and the fullscope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that Eosin Y does not affect cellular viability. HepG2 cells were treated for 24 hours with increasing concentrations (0.001to 100 μM) of Eosin Y, or left untreated (CTL). Staurosporine (STS) wasused as a positive control inducing cellular mortality. Higherconcentrations (0.5 and 1 mM) of Eosin Y could not be tested as the dyeinterfered with the assay.

FIG. 2 illustrates that Erythrosine B does not affect cellularviability. Hep G2 cells were treated for 24 hours with increasingconcentrations (0.001 to 100 μM) of Erythrosine B, or left untreated(CTL). Staurosporine (STS) was used as a positive control inducingcellular mortality. Higher concentrations (0.5 and 1 mM) of ErythrosineB could not be tested as the dye interfered with the assay.

FIG. 3 illustrates that initial wound closure is improved followingapplication of a wound healing composition. Rats (n=2 per group) with anexcision wound were treated or not with a wound healing compositioncomprising an oxidant (carbamide peroxide) and a photoactivator mixture(eosin Y, erythrosine B and Saffron Red Powder). Lozenges: treatedanimals; circles: untreated animals (controls).

FIG. 4 illustrates that wound closure is improved following applicationof wound healing compositions. Rats (n=2 per group) with an excisionwound were treated or not with wound healing composition (A) comprisingan oxidant (carbamide peroxide) and a photoactivator mixture (eosin Y,erythrosine B), wound healing composition (B) comprising an oxidant(carbamide peroxide) and a photoactivator mixture (eosin Y, erythrosineB and Saffron Red powder) or wound healing composition (C) comprising anoxidant (carbamide peroxide) and a photoactivator mixture (eosin Y,erythrosine B, Saffron Red powder and Indocyanin green).

FIG. 5 illustrates that wound closure is improved following applicationof a wound healing composition. Rats (n=2 per group) with an excisionwound were treated or not with a wound healing composition comprising anoxidant (carbamide peroxide) and a photoactivator mixture (eosin Y,erythrosine B). Lozenges: treated animals; triangles: untreated animals(controls).

FIG. 6 illustrates that wound closure is improved following applicationof a wound healing composition.

FIG. 7 illustrates that wound healing is improved following applicationof a wound healing composition.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with one embodiment, there is provided a wound healingcomposition and the method of use of the composition on a patient's skinor wound. This product accelerate healing and restoration of a wound.

In accordance with another embodiment, there is provided a method of useof the composition that is a photodynamic technique whereby thecomposition is activated by light, providing a beneficial effect on theskin or wound and promoting healing.

The composition and method may be used to treat injuries to thedifferent layers of the skin, including incisions, lacerations,abrasions, puncture wounds, penetrations wounds, gunshot wounds,contusions, hematomas and crushing injuries. Lesions to mucosae may alsobe treated with the composition of the present invention, which may beused for example, to treat pathological lesions of the oral mucosa, suchas periodontitis, ulcers, and cold sores (orofacial herpes).

The composition comprises a number of active principles selected fromgroups of possible components. These various active principles each havetheir mechanism of action.

Oxidants

The composition comprises oxidants as a source of oxygen radicals.Peroxide compounds are oxidants that contain the peroxy group (R—O—O—R),which is a chainlike structure containing two oxygen atoms, each ofwhich is bonded to the other and a radical or some element. Suitableoxidants for preparation of the active medium include, but are notlimited to:

Hydrogen peroxide (H₂O₂) is the starting material to prepare organicperoxides. H₂O₂ is a powerful oxidizing agent, and the unique propertyof hydrogen peroxide is that it breaks down into water and oxygen anddoes not form any persistent, toxic residual compound. Hydrogen peroxidefor use in this composition can be used in a gel, for example with 6%hydrogen peroxide. A suitable range of concentration over which hydrogenperoxide can be used in the present composition is from about 3.5% toabout 6%.

Urea hydrogen peroxide (also known as urea peroxide, carbamide peroxideor percarbamide) is soluble in water and contains approximately 35%hydrogen peroxide. Carbamide peroxide for use in this composition can beused as a gel, for example with 16% carbamide peroxide that represents5.6% hydrogen peroxide. A suitable range of concentration over whichurea peroxide can be used in the present composition is from about 10%to about 16%. Urea peroxide brakes down to urea and hydrogen peroxide ina slow-release fashion that can be accelerated with heat orphotochemical reactions. The released urea [carbamide, (NH₂)CO₂)], ishighly soluble in water and is a powerful protein denaturant. Itincreases solubility of some proteins and enhances rehydration of theskin and/or mucosa.

Benzoyl peroxide consists of two benzoyl groups (benzoic acid with the Hof the carboxylic acid removed) joined by a peroxide group. It is foundin treatments for acne, in concentrations varying from 2.5% to 10%. Thereleased peroxide groups are effective at killing bacteria. Benzoylperoxide also promotes skin turnover and clearing of pores, whichfurther contributes to decreasing bacterial counts and reduce acne.Benzoyl peroxide breaks down to benzoic acid and oxygen upon contactwith skin, neither of which are toxic. A suitable range of concentrationover which benzoyl peroxide can be used in the present composition isfrom about 2.5% to about 5%.

Inclusion of other forms of peroxides (e.g. organic or inorganicperoxides) should be avoided due to their increased toxicity and theirunpredictable reaction with the photodynamic energy transfer.

Photoactivators:

The photoactivators transfer light energy to the oxidants. Suitablephotoactivators can be fluorescent dyes (or stains), although other dyegroups or dyes (biological and histological dyes, food colorings,carotenoids) can also be used. Combining photoactivators may increasephoto-absorbtion by the combined dye molecules and enhance absorptionand photo-biomodulation selectivity. This creates multiple possibilitiesof generating new photosensitive, and/or selective photoactivatormixtures.

An advantageous characteristic of a photoactivator is increasedfluorescence. In the present invention, re-emission of light in thegreen to yellow spectrum would be advantageous, since it is a deeppenetrating wavelength range, with deep absorption by the blood. Thisconfers a strong increase on the blood flow, vasodilatation andangiokinetic phenomena. Suitable photoactivators include, but are notlimited to:

Xanthene Derivatives:

The xanthene derivative dyes have been used and tested for a long timeworldwide. They display low toxicity and increased fluorescence. Thexanthene group consists of 3 sub-groups that are: a) the fluorenes; b)fluorones; and c) the rhodoles.

The fluorenes group comprises the pyronines (e.g. pyronine Y and B) andthe rhodamines (e.g. rhodamine B, G and WT). Depending on theconcentration used, both pyronines and rhodamines may be toxic and theirinteraction with light may lead to increased toxicity. Similar effectsare known to occur for the rhodole dye group.

The fluorone group comprises the fluorescein dye and the fluoresceinderivatives.

Fluorescein is a fluorophore commonly used in microscopy with anabsorption max. of 494 nm and an emission max. of 521 nm. The disodiumsalt of fluorescein is known as D&C Yellow 8. It has very highfluorescence but photodegrades quickly. In the present composition,mixtures of fluorescein with other photoactivators such as indocyaningreen and/or saffron red powder will confer increased photoabsorption tothese other compounds.

Eosins group comprises Eosin Y (tetrabromofluorescein, acid red 87, D&CRed 22) with an abs. max 514-518 nm, stains cytoplasm of cells,collagen, muscle fibers and red blood cells intensely red; and Eosin B(acid red 91, eosin scarlet, dibromo-dinitrofluorescein), with the samestaining characteristics as Eosin Y. Eosin Y, eosin B, or a mixture ofboth can be used because of their sensitivity to the light spectra used:broad spectrum blue light, blue to green light and green light. Theirtissue and biofilm staining properties and their low toxicity are alsoadvantageous. Both eosin Y and eosin B stain red blood cells and thusconfer to the composition of the present invention haemostatic (controlsthe flow or stops the flow of blood) properties as well as increase theselective targeting of light to the soft tissues of the lesion or woundduring the application of the composition.

Phloxine B (2,4,5,7 tetrabromo 4,5,6,7,tetrachlorofluorescein, D&C Red28, acid red 92) is a red dye derivative of fluorescein which is usedfor disinfection and detoxification of waste water throughphotooxidation. It has an abs. max. of 535-548 nm. It is also used as anintermediate for making photosensitive dyes and drugs.

Erythrosine B (acid red 51, tetraiodofluorescein) is a cherry-pink,coal-based fluorine food dye used as a biological stain, and a biofilmand dental plaque disclosing agent, with max. abs. 524-530 nm in aqueoussolution. It is subject to photodegradation. Erythrosine is also used insome embodiments due to its photosensitivity to the light spectra usedand its ability to stain biofilms. Inclusion of erythrosine should befavored when using the composition in deep pockets of infected orcontaminated tissue, such as periodontal pockets in periodontal therapy.

Rose Bengal (4,5,6,7 tetrachloro 2,4,5,7 tetraiodofluorescein, acid red94) is a bright bluish-pink biological dye with an absorption max. of544-549 nm, that has been used as a dye, biological stain and diagnosticaid. Also used in synthetic chemistry to generate singlet from tripletoxygen.

Merbromine (mercurochrome) is an organo-mercuric disodium salt offluorescein with an abs. max. of 508 nm. It is used as an antiseptic.

Azo Dyes:

The azo (or diazo-) dyes share the N—N group, called azo the group. Theyare used mainly in analytical chemistry or as food colorings and are notfluorescent. Suitable azo dyes include: Methyl violet, neutral red, parared (pigment red 1), amaranth (Azorubine S), Carmoisine (azorubine, foodred 3, acid red 14), allura red AC (FD&C 40), tartrazine (FD&C Yellow5), orange G (acid orange 10), Ponceau 4R (food red 7), methyl red (acidred 2), murexide-ammonium purpurate.

Biological Stains:

Dye molecules commonly used in staining protocols for biologicalmaterials can also be used as photoactivators. Suitable biologicalstains include:

Saffranin (Saffranin 0, basic red 2) is also an azo-dye and is used inhistology and cytology. It Is a classic counter stain in a Gram stainprotocol.

Fuchsin (basic or acid) (rosaniline hydrochloride) is a magentabiological dye that can stain bacteria and has been used as anantiseptic. It has an abs. max. 540-555 nm.

3,3′ dihexylocarbocyanine iodide (DiOC6) is a fluorescent dye used forstaining cell's endoplasmic reticulum, vesicle membranes andmitochondria. It shows photodynamic toxicity; when exposed to bluelight, has a green fluorescence.

Carminic acid (acid red 4, natural red 4) is a red glucosidalhydroxyanthrapurin naturally obtained from cochineal insects.

Indocyanin green (ICG) is used as a diagnostic aid for blood volumedetermination, cardiac output, or hepatic function. ICG binds stronglyto red blood cells and when used in mixture with fluorescein, itincreases the absorption of blue to green light.

Carotenoids

Carotenoid dyes can also act as photoactivators.

Saffron red powder is a natural carotenoid-containing compound. Saffronis a spice derived from crocus sativus. It is characterized by a bittertaste and iodoform or hay-like fragrance; these are caused by thecompounds picrocrocin and saffranal. It also contains the carotenoid dyecrocin that gives its characteristic yellow-red color.

Saffron contains more than 150 different compounds many of them arecarotenoids: mangicrocin, reaxanthine, lycopene, and various α- andβ-carotenes, that show good absorption of light and beneficialbiological activity. Also saffron can act as both a photon-transferagent and a healing factor. Saffron color is primarily the result ofα-crocin (8,8 diapo-8,8-carotenoid acid). Dry saffron red powder ishighly sensitive to fluctuating pH levels and rapidly breaks downchemically in the presence of light and oxidizing agents. It is moreresistant to heat. Data show that saffron has anticarcinogenic,immunomodulating and antioxidant properties. For absorbance, it isdetermined for the crocin specific photon wavelength of 440 nm (bluelight). It has a deep red colour and forms crystals with a melting pointof 186° C. When dissolved in water it forms an orange solution.

Crocetin is another compound of saffron that was found to express anantilipidemic action and promote oxygen penetration in differenttissues. More specifically it was observed an increased oxygenation ofthe endothelial cells of the capillaries. An increase of the oxygenationof muscles and cerebral cortex was observed and led to an increasedsurvival rate in laboratory animals with induced hemorrhagic shock oremphysema.

Anatto a spice contains as main constituent (70-80%) the carotenoidbixin which displayed relevant antioxidative properties.

β-carotene, also displayed suitable characteristics.

Fucoxanthine is a constituent of brown algae with a pronounced abilityfor photosensitization of red-ox reactions.

Healing Factors:

Healing factors comprise compounds that promote or enhance the healingor regenerative process of the tissues on the application site of thecomposition. During the photoactivation of the composition, there is anincrease of the absorption of molecules at the treatment site by theskin or the mucosa. An augmentation in the blood flow at the site oftreatment is observed for an extent period of time. An increase in thelymphatic drainage and a possible change in the osmotic equilibrium dueto the dynamic interaction of the free radical cascades can be enhancedor even fortified with the inclusion of healing factors. Suitablehealing factors include, but are not limited to:

Hyaluronic acid (Hyaluronan, hyaluronate): is a non-sulfatedglycosaminoglycan, distributed widely throughout connective, epithelialand neural tissues. It is one of the primary components of theextracellular matrix, and contributes significantly to cellproliferation and migration. Hyaluronan is a major component of theskin, where it is involved in tissue repair. While it is abundant inextracellular matrices, it contributes to tissues hydrodynamics,movement and proliferation of cells and participates in a wide number ofcell surface receptor interactions, notably those including primaryreceptor CD44. The hyaluronidases enzymes degrade hyaluronan. There areat least seven types of hyaluronidase-like enzymes in humans, several ofwhich are tumor suppressors. The degradation products of hyaluronicacid, the oligosaccharides and the very-low molecular weight hyaluronicacid, exhibit pro-angiogenic properties. In addition, recent studiesshow that hyaluronan fragments, but not the native high molecular massof hyaluronan, can induce inflammatory responses in macrophages anddendritic cells in tissue injury. Hyaluronic acid is well suited tobiological applications targeting the skin. Due to its highbiocompatibility, it is used to stimulate tissue regeneration. Currentstudies evidenced hyaluronic acid appearing in the early stages ofhealing to physically create room for white blood cells that mediate theimmune response. It is used in the synthesis of biological scaffolds forwound healing applications and in wrinkle treatment.

Glucosamine: is one of the most abundant monosaccharides in humantissues and a precursor in the biological synthesis of glycosilatedproteins and lipids. It is commonly used in the treatment ofosteoarthritis. The common form of glucosamine used is its sulfate salt.Glucosamine shows a number of effects including an anti-inflammatoryactivity, stimulation of the synthesis of proteoglycans and thesynthesis of proteolytic enzymes. A suitable range of concentration overwhich glucosamine can be used in the present composition is from about1% to about 3%.

Allantoin: is a diureide of glyosilic acid. It has keratolytic effect,increases the water content of the extracellular matrix, enhances thedesquamation of the upper layers of dead (apoptotic) skin cells, andpromotes skin proliferation and wound healing.

Also, saffron can act as both a photon-transfer agent and a healingfactor.

Chelating Agents:

Chelating agents can be included to promote smear layer removal inclosed infected pockets and difficult to reach lesions; act as a metalion quencher and as a buffer. Suitable chelating agents include, but arenot limited to:

Ethylenediaminotetraacetic acid (EDTA): It is an aminoacid, used tosequester di- and trivalent metal ions. EDTA binds to metals via 4carboxylate and 2 amine groups. EDTA forms especially strong complexeswith Mn(III), Fe(III), Cu(III), Co(III). Prevents collection of theplatelets and blood clots formation. It is used in the endodontictherapy as a smear layer removal agent during instrumentation. It isused to buffer solutions.

Ethylene glycol tetraacetic acid (EGTA) is related to EDTA, but with amuch higher affinity for calcium than for magnesium ions. It is usefulfor making buffer solutions that resemble the environment inside livingcells and is often employed in dentistry, more specifically endodontics,in the removal of smear layer.

Lipolysis Stimulating Factors:

Lipolysis stimulating factors can be included for use of the compositionin cosmetic applications, such as wrinkle treatment.

Caffeine, and the metabolic derivative of caffeine paraxanthine canincrease in the lipolysis process to releases glycerol and fatty acidsinto the blood stream.

Hydrophilic Gelling Agents

The wound healing composition may also contain one or more hydrophilicgelling agent. The hydrophilic gelling agent enhances the consistency ofthe composition and contributes to facilitating the application of thecomposition to the skin or wounded area. Also, when used with hydrogenperoxide (H₂O₂), it may contribute to the slow the release of the H₂O₂,and provide a more immediate reaction because pure H₂O₂ can be useddirectly. Suitable hydrophilic gelling agent include, but are notlimited to glucose, modified starch, methyl cellulose, carboxymethylcellulose, propyl cellulose, hydroxypropyl cellulose, Carbopol®polymers, alginic acid, sodium alginate, potassium alginate, ammoniumalginate, calcium alginate, agar, carrageenan, locust bean gum, pectin,and gelatin.

Use of the Composition

The inclusion of suitable photosensitive compounds and activation with alight source of a proper wavelength, leads to the acceleration in thebreakdown process of the source of peroxide (the oxidant) and the otherreactions that take place, via a photodynamic phenomenon. The includeddyes are illuminated by photons of a certain wavelength and excited to ahigher energy state. When the photoactivators' excited electrons returnto a lower energy state, they emit photons with a lower energy level,thus causing the emission of light of a longer wavelength (Stokesshift). In the proper environment, much of this energy transfer istransferred to oxygen or the reactive hydrogen peroxide and causes theformation of oxygen radicals, such as singlet oxygen.

The singlet oxygen and other reactive oxygen species generated by theactivation of the composition are thought to operate in a hormeticfashion. That is, a health beneficial effect is brought about by the lowexposure to a normally toxic stimuli (e.g. reactive oxygen), bystimulating and modulating stress response pathways in cells of thetargeted tissues. Endogenous response to exogenous generated freeradicals (reactive oxygen species) is modulated in increased defensecapacity against the exogenous free radicals and induces acceleration ofhealing and regenerative processes. Furthermore, activation of thecomposition will also produce an antibacterial effect. The extremesensitivity of bacteria to exposure to free radicals makes thecomposition of the present invention a de facto bactericidalcomposition.

Possible mechanism of action should be a fortified redox signalingphenomenon resulting in accentuated signal transduction process in whichcells convert one kind of signal into another; activated “secondmessengers” induce a “signal cascade” beginning with a relatively smallstimulus that elicits a large response via biologically monitoredamplification of such signals. These complex mechanisms act possiblyinvolving angiogenic phenomena via growth factor activation.

This method could be described as a form of photodynamic therapy.However, unlike other photodynamic techniques, where thephotoactoactivators are incorporated in the tissue structure, in thepresent method, the photoactive material is in simple contact with thetissue and acts when activated by light, as a “photodynamic device” thatchemically interacts with the tissue. Additionally, the actinic lightpenetrates the tissue, and the light that is emitted by thephotoactivator (light of a longer wavelength) is also absorbed by thetissue.

Any source of actinic light can be used. Any type of halogen, LED orplasma arc lamp, or laser may be suitable. The primary characteristic ofsuitable sources of actinic light will be that they emit light in awavelength (or wavelengths) appropriate for activating the one or morephotoactivators present in the composition. In one embodiment, an argonlaser is used. In another embodiment, a potassium-titanyl phosphate(KTP) laser (e.g. a GreenLight™ laser) is used. In yet anotherembodiment, a LED photocuring device is the source of the actinic light.In yet another embodiment, the source of the actinic light is a sourceof visible light having a wavelength between 400 and 600 nm.Furthermore, the source of actinic light should have a suitable powerdensity. Suitable power density for non-collimated light sources (LED,halogen or plasma lamps) are in the range from about 900 mW/cm² to about2000 mW/cm². Suitable power density for laser light sources are in therange from about 0.5 mW/cm² to about 0.8 mW/cm².

The duration of the exposure to actinic light will be dependent on thesurface of the treated area, and on the type of lesion, trauma or injurythat is being treated. The photoactivation of the composition may takeplace within seconds or even fragment of seconds, but a prolongedexposure period is beneficial to exploit the synergistic effects of theabsorbed, reflected and reemitted light on the composition of thepresent invention and its interaction with the tissue being treated. Inone embodiment, the time of exposure to actinic light of the tissue,skin or wound on which the wound healing composition has been applied isa period between 60 second and 5 minutes. In another embodiment, thetime of exposure to actinic light of the tissue, skin or wound on whichthe wound healing composition has been applied is a period between 60seconds and 5 minutes per cm² of the area to be treated, so that thetotal time of exposure of a 10 cm² are would be between 10 minutes and50 minutes. In yet another embodiment, the source of actinic light is incontinuous motion over the treated area for the appropriate time ofexposure. In yet another embodiment, multiple applications of the woundhealing composition and actinic light are performed. In someembodiments, the tissue, skin or wound is exposed to actinic light atleast two, three, four, five or six times. In some embodiments, a freshapplication of the wound healing composition is applied before exposureto actinic light.

Alternative Embodiments Example I

An exemplary wound healing composition was prepared by mixing thefollowing components:

Oxidant Photoactivators Healing factor(s) Carbamide Erythrosine B (0.5%)Glucosamine peroxide (16%) Eosin B (0.25%) sulfate (3%) Saffron RedHyaluronic acid (3%) powder (0.25%)

The oxidant (4 mL) and healing factors (1.5 mL) were mixed and thecombined with the photoactivators (1 mL). The resulting composition wasapplied to the skin of a wounded patient, and activated with actiniclight provided by a LED photocuring device (blue light). The compositionwas removed following treatment.

Example II

An second exemplary wound healing composition was prepared by mixing thefollowing components:

Oxidant Photoactivators Healing factor(s) Carbamide FluoresceinGlucosamine peroxide (16%) Indocyanin green sulfate (3%) Saffron RedHyaluronic acid (3%) powder (0.25%)

The oxidant (4 mL) and healing factors (1.5 mL) were mixed and thecombined with the photoactivators (1 mL). The resulting composition wasapplied to the skin of, a wounded patient, and activated with actiniclight provided by a LED photocuring device (blue light). The compositionwas removed following treatment.

This second exemplary composition is using the fluorescein dye as aphotoactivator to other dyes (indocyanine green and saffron red powder)present in the composition. The addition of a small amount offluorescein to the indocyanine green and saffron red powder solutioncaused reemission of light at wavelengths that activated the other dyecompounds and improved the treatment by increasing the establishedclinical absorption/reemission criteria.

Indocyanine green binds well to hemoglobin and helps the selectiveenergy absorption by the tissues and also helps targeting these tissueswith the generated free radical cascades. Also, this photoactivatorsmixture is able to render saffron red fluorescent, which again improvesboth the photodynamic and biostimulating phenomena.

Example III

The toxicity of the photoactivators Eosin Y and Erythrosine B wasevaluated by measuring the cytotoxicity of these compounds on humancells. Hep G2 human hepatocellular carcinoma cells with an epithelialmorphology were treated for 24 hours with increasing concentrations(0.001 to 100 μM) of Eosin Y or Erythrosine B, and the cellular survivalwas evaluated. Increasing concentrations of either Eosin Y (FIG. 1) orErythrosine B (FIG. 2) did not affect cellular viability when comparedto untreated cells. Staurosporine (STS) was used as a positive controlfor inducing cellular mortality and caused a dose-dependent effect(FIGS. 1 and 2). Similar results were obtained by measuring cell deathby release of lactate dehydrogenase (LDH). Therefore, neither Eosin Y orErythrosine B caused increased cellular mortality.

Example IV Excisional Wound model in Rat

Random skin flaps in rat were used to study the wound healingprocedures, for evaluating the benefits of ischemic and pharmacologicpreconditioning methods on skin flap survival, applying blood flowassessment technologies on flaps, demonstrating the effects of vascularshunts and the studies on skin flap viability. The random skin flapmodel was used to study the effect of the composition of the presentinvention on skin flap survival and associated modulations contributingto healing process.

Excision wound of 1 cm in width by 2 cm in length were cut dorsally onthe midline of the back, 2 cm below the inferior angle of the scapulae.The skin was cut with a surgical blade, the panniculus carnosus and a0.5 cm layer subcutaneous to the panniculus carnosus was excised fromthe wound edges. The wound was next photographed with an 8 mm by 8 mmsize marker. One gram of the wound healing composition was applied tothe wound (0.5 g/cm²) and irradiated with a blue LED light during 3minutes.

Example V

Excisions were performed on rats (n=2 per group) as described above inExample IV, and the excisions were treated or not with a singleapplication of 1 gram of a wound healing composition comprising theoxidant (carbamide peroxide) and a photoactivator mixture containingeosin Y, erythrosine B and saffron red powder. The excisions wereirradiated with a LED light (blue light) for 3 minutes. The percentageof wound closure was evaluated (FIG. 3) over a ten-day period followingtreatment. Animals treated with the composition showed a more rapidwound closure over the initial first three-day period followingtreatment.

Example VI

Excision were performed on rats (n=2 per group) as described above inExample IV, and the excisions were treated or not with a singleapplication of 1 gram of a wound healing compositions comprising: (A)the oxidant (carbamide peroxide) and a photoactivator mixture containingeosin Y, and erythrosine B; (B) the oxidant (carbamide peroxide) and aphotoactivator mixture containing eosin Y, erythrosine B and saffron redpowder; or (C) the oxidant (carbamide peroxide) and a photoactivatormixture containing eosin Y, erythrosine B, saffron red powder andindocyanine green. The excisions were irradiated with a LED light (bluelight) for 3 minutes. The percentage of wound closure was evaluateddaily for four days (FIG. 4). Animals treated with compositions (A) and(B) showed improved wound closure over the four-day period followingtreatment. The addition of indocyanine green in composition (C)repressed the wound healing effect observed for compositions (A) and(B).

Example VII

Excision were performed on rats (n=2 per group) as described above inExample IV, and the excisions were treated or not with a singleapplication of 1 gram of a wound healing composition comprising theoxidant (carbamide peroxide) and a photoactivator mixture containingeosin Y and erythrosine B. The excisions were irradiated with a LEDlight (blue light) for 3 minutes. The percentage of wound closure wasevaluated (FIG. 5) over a twelve-day period. Animals treated with thecomposition showed a more rapid wound closure over the first seven-dayperiod following treatment.

Example VIII

Using a template of 3 cm by 9 cm (3×9) in plexiglas, a flap was tracedon the dorsal skin with a surgical marker, taking as limits the inferiorangles of the scapulae and the superior bones of pelvis. A pure randompattern flap with cranial base was cut using sterile techniques andelevated through deep fascia, including superficial fascia, panniculuscarnosus, the subcutaneous tissue and skin. To minimize woundcontraction and simulating the human condition, a 0.5 cm subcutaneouslayer of panniculus carnosus from the wound edges was removed. Duringone hour, an impermeable barrier (e.g. a silicone sheeting) was placedbetween the flap and its donor site to eliminate the possibility ofwound bed support. The sheet was then removed, the flap returned to itsoriginal position and the flap edges were surgically closed using 4/0nylon suture in an interrupted fashion. Immediately following flapclosure, flap pedicle were coated with 13.5 g of gel formulation (0.5g/cm²) and irradiated. The controls did not receive any treatment. Carewas taken to distribute ointment evenly along the entire flap. Gelformulation was prepared the same day of the experiment. For Gel+Lightgroup the animals were treated with gel formulation, the flap wasirradiated for 3 minutes with a LED lamp.

Example IX

Excision were performed on rats (n=2 per group) as described above, andthe excisions were treated or not with the gel formulation, andirradiated with a LED light (blue light) as described in Example VIII.

The results demonstrate a direct correlation of necrosis fromfluorescein injection and direct visualization. Biopsies were evaluatedfor changes in histology. Data from the treated group demonstrate aclinically significant 1.5 times reduction in necrosis, (percentnecrosis, mean, SD of 45.7 (±17.36) vs. 30.42 (±20.18), in the controland treatment groups respectively). Now referring to FIG. 7A, theclinical evaluation of necrosis following flap surgery, in the controland treatment group shows that higher necrosis is observed on thecontrol group versus the treatment group.

Hematoxylin and eosin staining of biopsies from the control andtreatment group (FIG. 7B) reveal that greater vascular recruitmentoccurs in the treated group (see black arrows therein). Masson Trichromestaining for the assessment of collagen fibril deposition (FIG. 7C) at a40× magnification shows that new collagen deposition is occurring in thetreatment group vs the control group. The photodynamic treatment usingphotoactivators and wavelength specific light aimed at increasing theviability of the skin flap by stimulating the vascular recruitment ofcollaterals in flaps to improve the loco-regional state of the newwound, including the formation of new collagen therein.

The embodiments and examples presented herein are illustrative of thegeneral nature of the subject matter claimed and are not limiting. Itwill be understood by those skilled in the art how these embodiments canbe readily modified and/or adapted for various applications and invarious ways without departing from the spirit and scope of the subjectmatter disclosed claimed. The claims hereof are to be understood toinclude without limitation all alternative embodiments and equivalentsof the subject matter hereof. Phrases, words and terms employed hereinare illustrative and are not limiting. Where permissible by law, allreferences cited herein are incorporated by reference in their entirety.It will be appreciated that any aspects of the different embodimentsdisclosed herein may be combined in a range of possible alternativeembodiments, and alternative combinations of features, all of whichvaried combinations of features are to be understood to form a part ofthe subject matter claimed.

The invention claimed is:
 1. A method for wound healing, comprising: (a)topically applying on a patient's wound an effective amount of acomposition comprising at least one oxidant, a fluorescent compound, anda healing factor selected from hyaluronic acid, glucosamine andallantoin; and (b) exposing said composition to actinic light having anemission wavelength between about 400 nm and 600 nm for a timesufficient to cause a change in color of the fluorescent compound. 2.The method according to claim 1, wherein said composition is exposed toactinic light for a period of about 60 seconds to about 5 minutes. 3.The method according to claim 1, wherein said composition is exposed toactinic light for a period of about 60 seconds to about 5 minutes percm² of an area to be treated.
 4. The method according to claim 1,wherein the source of actinic light is in continuous motion over an areato be treated.
 5. The method according to claim 1, wherein the oxidantis selected from hydrogen peroxide, carbamide peroxide and benzoylperoxide.
 6. The method according to claim 1, wherein the compositionfurther comprises at least one hydrophilic gelling agent.
 7. The methodaccording to claim 6, wherein the hydrophilic gelling agent is selectedfrom glucose, modified starch, methyl cellulose, carboxymethylcellulose, propyl cellulose, hydroxypropyl cellulose, carbomer polymers,alginic acid, sodium alginate, potassium alginate, ammonium alginate,calcium alginate, agar, carrageenan, locust bean gum, pectin, andgelatin.
 8. The method according to claim 1, wherein the fluorescentcompound is Eosin Y.
 9. The method according to claim 1, wherein thefluorescent compound is selected from a xanthene derivative dye, an azodye, a biological stain and a carotenoid.
 10. The method according toclaim 1, wherein the composition further comprises at least one ofrhodamine B, rhodamine WT, rhodamine G, phloxine B, rose bengal, eosinB, fluorescein, erythrosine B, saffranin O, basic fuchsin, acid fuschin,3,3′ dihexylocarbocyanine iodide, carminic acid, indocyanine green,crocetin, α-crocin (8,8-diapo-8,8-carotenoic acid), zeaxanthine,lycopene, α-carotene, β-carotene, merbromine, bixin, fucoxanthine,methyl violet, neutral red, para red, amaranth, carmoisine, allura redAC, tartrazine, orange G, ponceau 4R, methyl red, murexide-ammoniumpurpurate, pyronine Y and pyronine B.
 11. The method of claim 1, whereinthe wound is an injury of the skin selected from incisions, lacerations,abrasions, puncture wounds, penetrations wounds, gunshot wounds,contusions, hematomas and crushing injuries.
 12. The method of claim 1,wherein the wound is a lesion of the oral mucosa selected fromperiodontitis, oral ulcers, and cold sores.
 13. The method of claim 1,wherein the wound is exposed to the actinic light at least two, three,four, five or six times, and wherein a fresh application of thecomposition is applied before exposure to the actinic light.